We previously re-engineered the anthrax toxin proteins to produce a highly specific anti-angiogenic cancer therapeutic, able to strongly repress tumor growth in mouse models. This was achieved by modifying protective antigen (PA) so that its activation and toxicity require the presence of two proteases, matrix metalloproteinase (MMP) and urokinase plasminogen activator (uPA). These two proteases are upregulated in tumor microenvironments. Thus, the therapeutic agent consists of two PA variants, which are individually nontoxic, but form functional toxins upon complementary oligomerization. These PA variants together deliver a toxic effector, usually anthrax lethal factor (LF). Because the use of three proteins (the two PA proteins and LF) would complicate the regulatory process, we sought to reduce the agent to a mixture of two components. Thus, in this FY2019, we created a dual-protease PA targeting system which utilizes bismaleimide cross-linked PA (CLPA) rather than the intercomplementing PA variants. Three different CLPA agents were tested and, as expected, found to exclusively form octamers. Two of these had in vitro toxicities equal to those of previous intercomplementing agents. Overall, this work advances the development and use of the PA and LF tumor-targeting system as a practical cancer therapeutic, as it provides a way to reduce the drug components of the anthrax toxin drug delivery system from three to two, which may lower the cost and simplify testing in clinical trials. As part of the effort to develop improved vaccines for biothreat agents, we worked with collaborators at Catholic University to use bacteriophage T4 particles as a platform for display and presentation of antigens of both Bacillus anthracis and Yersinia pestis, the latter being the causative agent of plague. The surface of the capsid head of T4 phage has a small, non-essential but tightly bound accessory protein called Soc. Capsids produced without Soc can be mixed with recombinant protein antigens produced as fusions to Soc. In the present case, Soc fusions were made to anthrax PA, and to two plague antigens - a mutated form of capsular antigen F1 and the low-calcium-response V antigen of the type 3 secretion system. The nanoparticles produced by mixing these fusion proteins with the Soc-deficient capsids elicited robust anthrax- and plague-specific immune responses and provided complete protection against inhalational anthrax and/or pneumonic plague in three animal challenge models, namely, mice, rats, and rabbits. Protection was demonstrated even when the animals were simultaneously challenged with lethal doses of both anthrax lethal toxin and Y. pestis bacteria. Unlike traditional subunit vaccines, the phage T4 vaccine uses a highly stable nanoparticle scaffold, provides multivalency, requires no adjuvant, and elicits broad T-helper 1 and 2 immune responses that are essential for complete clearance of bacteria during infection. This phage T4-based vaccine is a unique vaccine against two high-risk pathogens. It also illustrates a promising approach to generating vaccines against multiple pathogens. The lesser-studied component of anthrax toxin is edema factor (EF), a calmodulin- and calcium-dependent adenylyl cyclase. This enzyme is highly active. In cultured cells it can produce large amounts of cAMP that far exceed those needed to activate downstream pathways, and in some cases it can even substantially deplete cellular levels of its substrate, ATP. In work reported in FY2019, our collaborators at CDC have exploited the high catalytic activity of EF to develop a highly sensitive assay for EF. This three-step method includes magnetic immunocapture of EF with monoclonal antibodies, reaction with ATP to generate cAMP, and quantification of the cAMP by isotope-dilution HPLC-MS/MS. The detection limit was 20 fg/mL (225 zeptomoles/ml). The assay demonstrated 100% sensitivity in samples from 3 human and 5 rhesus macaques with inhalation anthrax. Analysis of EF in the rhesus macaques showed that it was detected earlier post-exposure than B. anthracis by culture and PCR. Thus, this assay has the potential to diagnosis anthrax infection at very early stages, when antibiotic administration has the greatest chance of being curative. A unique feature of the anthrax toxin proteins is the assembly of a PA protein oligomer that, once internalized to endosomea, inserts into the lipid bilayer to produce a protein-conducting channel. This structure is very well studied and characterized, with 3D structures obtained, and extensive mutagenesis done to define the roles of many key amino acid residues. Less well understood is the dependence of this channel on the composition of the lipid bilayer that surrounds it. Fortunately, sophisticated electrophysiological techniques exist for study of such channels. In particular, it is possible to insert single channels into artificial lipid bilayers and measure their electrical behavior in detail. In the current reporting period, our collaborators at Catholic University have examined the effect of varying the lipid composition of the bilayer. This is relevant in part because the membrane lipid composition of early and late endosomes differ. Using the model bilayer system, it was found that membrane lipids can have a strong effect on the anthrax toxin channel properties, including the channel-forming activity, voltage-gating, conductance, selectivity, and enzymatic factor binding. Interestingly, the highest channel insertion rate was observed in bis(monoacylglycero)phosphate-containing membranes. A molecular dynamics simulation showed that the conformational properties of the channel are different in bis(monoacylglycero)phosphate compared to phosphatidyl-choline, phosphatidyl-serine, and phosphatidyl-ethanolamine lipids.